Takashi Okubo

Publications of Takashi Okubo

• Pyrosequence Read Length of 16S rRNA Gene Affects Phylogenetic Assignment of Plant-associated Bacteria.

  Authors: Takashi Okubo, Seishi Ikeda, Akifumi Yamashita, Kimihiro Terasawa, Kiwamu Minamisawa

  Microbes and environments / JSME. 02/2012;

  Pyrosequence targeting of the 16S rRNA gene has been adopted for microbial communities associated with field-grown plants. To examine phylogenetic drifts according to read length and bioinformaticPyrosequence targeting of the 16S rRNA gene has been adopted for microbial communities associated with field-grown plants. To examine phylogenetic drifts according to read length and bioinformatic tools, original and chopped sequences (250-570 bp) covering the V1-V4 regions of 16S rRNA genes were compared using pyrosequence and Sanger reads of rice root microbiomes. The phylogenetic assignment at genus level depended on read length, especially in the genus Bradyrhizobium, which is one of the ecologically important bacterial genera associated with plants. We discuss the methodology of phylogenetic assignments of plant-associated bacteria by 16S rRNA pyrosequence.

• The genotype of the calcium/calmodulin-dependent protein kinase gene (CCaMK) determines bacterial community diversity in rice roots under paddy and upland field conditions.

  Authors: Seishi Ikeda, Takashi Okubo, Naoya Takeda, Mari Banba, Kazuhiro Sasaki, Haruko Imaizumi-Anraku, Shinsuke Fujihara, Yoshinari Ohwaki, Kenshiro Ohshima, Yoshimichi Fukuta, Shima Eda, Hisayuki Mitsui, Masahira Hattori, Tadashi Sato, Takuro Shinano, Kiwamu Minamisawa

  Applied and environmental microbiology. 07/2011; 77(13):4399-405.

  The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [R]) on rice root-associated bacteria,The effects of the Oryza sativa calcium/calmodulin-dependent protein kinase OsCCaMK genotype (dominant homozygous [D], heterozygous [H], recessive homozygous [R]) on rice root-associated bacteria, including endophytes and epiphytes, were examined by using a Tos17 rice mutant line under paddy and upland field conditions. Roots were sampled at the flowering stage and were subjected to clone library analyses. The relative abundance of Alphaproteobacteria was noticeably decreased in R plants under both paddy and upland conditions (0.8% and 3.0%, respectively) relative to those in D plants (10.3% and 17.4%, respectively). Population shifts of the Sphingomonadales and Rhizobiales were mainly responsible for this low abundance in R plants. The abundance of Anaerolineae (Chloroflexi) and Clostridia (Firmicutes) was increased in R plants under paddy conditions. The abundance of a subpopulation of Actinobacteria (Saccharothrix spp. and unclassified Actinosynnemataceae) was increased in R plants under upland conditions. Principal coordinate analysis revealed unidirectional community shifts in relation to OsCCaMK gene dosage under both conditions. In addition, shoot length, tiller number, and plant weight decreased as the OsCCaMK gene dosage decreased under upland conditions. These results suggest significant impacts of OsCCaMK on both the diversity of root-associated bacteria and rice plant growth under both paddy and upland field conditions.

• Isolation and genetic characterization of Aurantimonas and Methylobacterium strains from stems of hypernodulated soybeans.

  Authors: Mizue Anda, Seishi Ikeda, Shima Eda, Takashi Okubo, Shusei Sato, Satoshi Tabata, Hisayuki Mitsui, Kiwamu Minamisawa

  Microbes and environments / JSME. 06/2011; 26(2):172-80.

  The aims of this study were to isolate Aurantimonas and Methylobacterium strains that responded to soybean nodulation phenotypes and nitrogen fertilization rates in a previous culture-independentThe aims of this study were to isolate Aurantimonas and Methylobacterium strains that responded to soybean nodulation phenotypes and nitrogen fertilization rates in a previous culture-independent analysis (Ikeda et al. ISME J. 4:315-326, 2010). Two strategies were adopted for isolation from enriched bacterial cells prepared from stems of field-grown, hypernodulated soybeans: PCR-assisted isolation for Aurantimonas and selective cultivation for Methylobacterium. Thirteen of 768 isolates cultivated on Nutrient Agar medium were identified as Aurantimonas by colony PCR specific for Aurantimonas and 16S rRNA gene sequencing. Meanwhile, among 187 isolates on methanol-containing agar media, 126 were identified by 16S rRNA gene sequences as Methylobacterium. A clustering analysis (>99% identity) of the 16S rRNA gene sequences for the combined datasets of the present and previous studies revealed 4 and 8 operational taxonomic units (OTUs) for Aurantimonas and Methylobacterium, respectively, and showed the successful isolation of target bacteria for these two groups. ERIC- and BOX-PCR showed the genomic uniformity of the target isolates. In addition, phylogenetic analyses of Aurantimonas revealed a phyllosphere-specific cluster in the genus. The isolates obtained in the present study will be useful for revealing unknown legume-microbe interactions in relation to the autoregulation of nodulation.

• Community- and genome-based views of plant-associated bacteria: plant-bacterial interactions in soybean and rice.

  Authors: Seishi Ikeda, Takashi Okubo, Mizue Anda, Hideo Nakashita, Michiko Yasuda, Shusei Sato, Takakazu Kaneko, Satoshi Tabata, Shima Eda, Ayumi Momiyama, Kimihiro Terasawa, Hisayuki Mitsui, Kiwamu Minamisawa

  Plant & cell physiology. 09/2010; 51(9):1398-410.

  Diverse microorganisms are living as endophytes in plant tissues and as epiphytes on plant surfaces in nature. Questions about driving forces shaping the microbial community associated with plantsDiverse microorganisms are living as endophytes in plant tissues and as epiphytes on plant surfaces in nature. Questions about driving forces shaping the microbial community associated with plants remain unanswered. Because legumes developed systems to attain endosymbioses with rhizobia as well as mycorrhizae during their evolution, the above questions can be addressed using legume mutants relevant to genes for symbiosis. Analytical methods for the microbial community have recently been advanced by enrichment procedures of plant-associated microbes and culture-independent analyses targeting the small subunit of rRNA in microbial ecology. In this review, we first deal with interdisciplinary works on the global diversity of bacteria associated with field-grown soybeans with different nodulation genotypes and nitrogen application. A subpopulation of Proteobacteria in aerial parts of soybean shoots was likely to be regulated through both the autoregulation system for plant-rhizobium symbiosis and the nitrogen signaling pathway, suggesting that legumes accommodate a taxonomically characteristic microbial community through unknown plant-microbe communications. In addition to the community views, we then show multiphasic analysis of a beneficial rice endophyte for comparative bacterial genomics and plant responses. The significance and perspectives of community- and genome-based approaches are discussed to achieve a better understanding of plant-microbe interactions.

• Community shifts of soybean stem-associated bacteria responding to different nodulation phenotypes and N levels.

  Authors: Seishi Ikeda, Takashi Okubo, Takakazu Kaneko, Shoko Inaba, Tomiya Maekawa, Shima Eda, Shusei Sato, Satoshi Tabata, Hisayuki Mitsui, Kiwamu Minamisawa

  The ISME journal. 11/2009;

  The diversity of stem-associated bacteria of non-nodulated (Nod(-)), wild-type nodulated (Nod(+)) and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S ribosomalThe diversity of stem-associated bacteria of non-nodulated (Nod(-)), wild-type nodulated (Nod(+)) and hypernodulated (Nod(++)) soybeans were evaluated by clone library analyses of the 16S ribosomal RNA gene. Soybeans were dressed with standard nitrogen (SN) fertilization (15 kg N ha(-1)) and heavy nitrogen (HN) fertilization (615 kg N ha(-1)). The relative abundance of Alphaproteobacteria in Nod(+) soybeans (66%) was smaller than that in Nod(-) and Nod(++) soybeans (75-76%) under SN fertilization, whereas that of Gammaproteobacteria showed the opposite pattern (23% in Nod(+) and 12-16% in Nod(-) and Nod(++) soybeans). Principal coordinate analysis showed that the bacterial communities of Nod(-) and Nod(++) soybeans were more similar to each other than to that of Nod(+) soybeans under SN fertilization. HN fertilization increased the relative abundance of Gammaproteobacteria in all nodulation phenotypes (33-57%) and caused drastic shifts of the bacterial community. The clustering analyses identified a subset of operational taxonomic units (OTUs) at the species level in Alpha- and Gammaproteobacteria responding to both the nodulation phenotypes and nitrogen fertilization levels. Meanwhile, the abundance of Betaproteobacteria was relatively constant in all libraries constructed under these environmental conditions. The relative abundances of two OTUs in Alphaproteobacteria (Aurantimonas sp. and Methylobacterium sp.) were especially sensitive to nodulation phenotype and were drastically decreased under HN fertilization. These results suggested that a subpopulation of proteobacteria in soybeans is controlled in a similar manner through both the regulation systems of plant-rhizobia symbiosis and the nitrogen signaling pathway in plants.The ISME Journal advance online publication, 19 November 2009; doi:10.1038/ismej.2009.119.

• Development of a Bacterial Cell Enrichment Method and its Application to the Community Analysis in Soybean Stems.

  Authors: Seishi Ikeda, Takakazu Kaneko, Takashi Okubo, Lynn Rallos, Shima Eda, Hisayuki Mitsui, Shusei Sato, Yasukazu Nakamura, Satoshi Tabata, Kiwamu Minamisawa

  Microbial ecology. 09/2009;

  A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. StemA method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.

• Nodulation-dependent communities of culturable bacterial endophytes from stems of field-grown soybeans.

  Authors: Takashi Okubo, Seishi Ikeda, Takakazu Kaneko, Shima Eda, Hisayuki Mitsui, Shusei Sato, Satoshi Tabata, Kiwamu Minamisawa

  Microbes and environments / JSME. 01/2009; 24(3):253-8.

  Endophytic bacteria (247 isolates) were randomly isolated from surface-sterilized stems of non-nodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans (Glycine maxEndophytic bacteria (247 isolates) were randomly isolated from surface-sterilized stems of non-nodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans (Glycine max [L.] Merr) on three agar media (R2A, nutrient agar, and potato dextrose agar). Their diversity was compared on the basis of 16S rRNA gene sequences. The phylogenetic composition depended on the soybean nodulation phenotype, although diversity indexes were not correlated with nodulation phenotype. The most abundant phylum throughout soybean lines tested was Proteobacteria (58-79%). Gammaproteobacteria was the dominant class (21-72%) with a group of Pseudomonas sp. significantly abundant in Nod(+) soybeans. A high abundance of Alphaproteobacteria was observed in Nod(-) soybeans, which was explained by the increase in bacterial isolates of the families Rhizobiaceae and Sphingomonadaceae. A far greater abundance of Firmicutes was observed in Nod(-) and Nod(++) mutant soybeans than in Nod(+) soybeans. An impact of culture media on the diversity of isolated endophytic bacteria was also observed: The highest diversity indexes were obtained on the R2A medium, which enabled us to access Alphaproteobacteria and other phyla more frequently. The above results indicated that the extent of nodulation changes the phylogenetic composition of culturable bacterial endophytes in soybean stems.

• Microbial community analysis of field-grown soybeans with different nodulation phenotypes.

  Authors: Seishi Ikeda, Lynn Esther E. Rallos, Takashi Okubo, Shima Eda, Shoko Inaba, Hisayuki Mitsui, Kiwamu Minamisawa

  Applied and environmental microbiology. 09/2008; 74(18):5704-9.

  Microorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomalMicroorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod(+) soybean roots. Fusarium solani was stably associated with nodulated (Nod(+) and Nod(++)) roots and less abundant in Nod(-) soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod(-), Nod(+), or Nod(++)). The analysis of root samples indicated that the microbial community in Nod(-) soybeans was more similar to that in Nod(++) soybeans than to that in Nod(+) soybeans.